( Reaffirmed 2001 ) IS : 11383- 1985 Indian Standard SPECIFICATION FOR THIN WALLED GLASS CAPILLARY PIPETTES Medical Glass Instruments and Appliances CPDC 12 Sectional Committee, Chairman DR S. SRI~AMAC~~RI Members &RI Representiqg Indian Council of Medical Research, New Delhi Ministry of Defence ( DGI ) V. K. BAJPU SRRI J. N. VISU~ARAR~~A ( Alternate ) Directorate of Medical Education & Research, DR S. G. DEODHARE Bombay Hindustan Antibiotics Ltd, Punt DR I. S. GANDHI Directorate General of Armed Forces Medical BRIG M. I. HASSAN Services ( Ministry of Defence ), New Delhi National Phvsical Laboratorv , ( DR K. C. .Toslrr . CSIR ). New Delhi SHRI P: C. JTAIN ( Alternate ) Scientific Glass Equipment Manufacturer's DR K. C. KRERA Association, llombay SHRI V. V. KETIUI~ ( Alternate ) Railway Board ( Ministrv of Railways ), New Delhi DR G. D. MODY SHRI R. N. MUKITERJEE Rohem'Instruments Private Limited,`N&ik SR~ZI M. K. DUTTA ( Alternate ) Steriware (P) Ltd, Faridabad SHRI RAJIV MADAN SHRI A. J ALES ( Alternate ) Borosil Glass Works Ltd, Bombay SHRI P. R. Rao SERI A. K. BANSAL ( Alternate j Haffkine Bio-Pharmaceutical Corporation Ltd, DR F. L. SALDRALA Born hay SHRI A. B. CRUNOIIKAR ( Allernate) Scientific Indian Glass Co Ltd, Calcutta SHRI SARUP SIRCAR SHRI N. G. SIRCAR C Alternate ) ' Top Syringe Manufacturing Co, Bombay SIIRI J. C. SIIANDILYA ~ SHRI J. K. WAD ( Alternate ) Directorate General of Health Services ( Ministry of DR R. C. SI'IARMA Health & Family Welfare j, New Delhi All India Instrument Manufacturers' and Dealers' SHRI Y. D. SHARM`Z Association. New Delhi SHRI P. N. SOOD ( Alternate I ) SRRI J. C. SHA~DILYA ( Alternate II ) ( Continued on pqe 2 ) @ Copyright 1986 INDIAN STANDARDS INSTITUTION This publication is protected under the Indian Copyright Act ( XIV of 1957 ) and reproduction in whole or in part by any means except with written permission of the publisher shall be deemed to be an infringement of copyright under the said Act_ IS:11383 - 1985 (Continued from page 1 1 Rekresenting Central Glass & Ceramic Research Institute ( CSIR \_Calcutta Directorate `beneral of Technical Development, New Delhi Directorate General of Health Services ( Ministry of Health and Family Welfare ), New Delhi Director General, IS1 ( Ex-ojicio Member ) Members DR K. P. SRIVASTATA SHRI R. SHRI SOUNDHIRARAJAN B. N. DAS ( Alternate ) RANI & Med DR SUDHA DR A. S. SETHI, Director ( Consr Prod Instrs ) Secretal;v Joint Director SHRI D. K. AQRAWAL ( Consr Prod & Med Instrs ), ISI Glass Instruments Convener COLS.CRAERAV~RTY and Appliances Subcommittee, CPDC 12 : 2 Directorate General of Armed Forces Medical Services ( Ministry of Defence ), New Delhi Ministry of Defence ( DGI ) MemCers SHRI V. K. BAJPAI SHRI G. TRIVEDI ( Alternate ) SRRI HARI DUTTA DR(SNIT)R.GUPTA Ganga Glass Works PVC Ltd, Balawali Directorate General of Health Services ( Ministry of Health & Family Welfare ), New Delhi Borosil Glass Works Limited, Bombay SHRI P. R. RAO Haffkine Bio-Pharmaceutical Corporation Limited, DRF.L. SALDEANA Bombay SHIXIA. B. CHUNODEAR ( Ahnate ) Scientific Indian Glass Co Ltd, Calcutta SHRI SHRUB SI~CAR SHRI N. G. SIRC~R ( Alternate ) Top Syringe Manufacturing Co, Bombay SRRIJ.C.SHANDILYA SHRI J. K. WAD ( Alternate ) 2 IS :11383 - 1985 Indian Standard SPECIFICATION FOR THIN WALLED GLASS CAPILLARY PIPETTES 0. FOREWORD 0.1 This Indian Standard was adopted by the Indian Standards Institution on 16 September 1985, after the draft finalized by the Medical Glass Instruments and Appliances Sectional Committee had been approved by the Consumer Products and Medical Instruments Division Council. 0.2 Originally thin walled glass capillary pipettes were introduced for microhaematocrit procedures and even today they are used in biological and medical sciences predominantly, but with advancement of science and technology they have gained wide spread acceptance in many other fields of science where micro-technique works are involved. 0.3 The thin walled glass capillary pipettes are generally used as disposable one. For practical purpose the word `disposable' expressed in this standard and described in these pipettes means `one time use only'. A caution note should also be added as `any individual or institution who reuses these pipettes must bear full responsibility for its safety and effectiveness'. 0.4 Assistance standards: has been derived from the following Principles Pipettes of international design and IS0 384-1978 construction IS0 1769-1975 Laboratory glass wares of volumetric glassware Laboratory glass ware - Colour coding 0.5 For the purpose of deciding whether a particular requirement of this standard is complied with, the final value, observed or calculated, expressing the result of a test or analysis, shall be rounded off in accordance with IS : 2-1960*. The number of significant places retained in the rounded off value should be the same as that of the specified value in this standard. *Rules for rounding off numerical value ( revised ). 3 IS : 11383- 1985 1. SCOPE 1.1 This standard specifies general requirements and test for thin walled glass capillary pipettes used generally in haematological and biological work. 1.2 These pipettes which are heparinized, `one time use only' for delivering micro methods given in the standard. 2. CLASSIFICATION 2.1 This standard covers the following types of pipettes: a) QPe 1 b) Type2 Where dimensions are necessary adjunct without reference to volumetric capacity. Where specific volumetric capacity is necessary adjunct when filled end to end. siliconized quantities or plain are for of liquid as per c) TYPC3 3. REFERENCE Where specific volumetric capacity is necessary adjunct when filled up to ring mark(s) graduation. TEMPERATURE - 3.1 The standard reference temperature, that is, temperature at which the pipette is intended to contain its nominal volume ( nominal capacity ) is 27°C. 4. UNIT OF VOLUME ( mm3) for which the name 4.1 The unit of volume is cubic millimetre microlitre .may be used. NOTE millimetre The term microlitre ( ~1 ) is commonly used as a special name for cubic ( mm0 ) in accordance with International System of Units ( SI ). 5. MATERIALS 5.1 Glass - The pipette shall be manufactured properties as required for the end use. from glass of suitable 5.2 Heparin - Heparin used for coating the inner surface of the pipette shall be either sodium, lithiumor ammonium salt as required for end use. 5.2.1 When sodium or lithium salt heparin is used for coating the inner surface of the pipette, the heparin potency shall be 100 USP units per milligram of sodium or lithium salt heparin used. 5.2.2 When ammonium salt heparin is used for coating surface of the pipette, the heparin potency shall be 60 USP milligram of ammonium salt heparin used. 4 the inner units per IS :11383 6. DESIGN, CONSTRUCTION AND WORKMANSHIP - 1985 6.1 Construction of pipette shall be one straight piece, open at both The design dimensions and perends, without lips or construction. missible variations shall be in accordance with respective tables and figures. 6.2 Any cross-section of the pipette taken in a plane longitudinal axis shall preferably be circular. perpendicular to the 6.3 The pipette shall be free from defects that noticeably detract from their appearance or impair their serviceability. The pipette shall be free from foreign matter, loose or embedded line or chips that effect the bore or stains when viewed under normal room lighting. 6.4 The pipette ends shall be cut approximately at 90" to the longitudinal axis and shall not be cracked or have jagged ends or chips that enter the bore of the pipette. 6.5 6.6 Ends of the pipette may be fire polished. Ring Mark/Marks 6.6.1 a) Ring mark for colour coding ( that is, volumetric capacity to contain in the pipette to which the liquid identification ). to contain of b) Ring mark/marks up volumetric capacity. 6.6.2 Ring marks for deposited on the glass capacity in accordance in accordance with the normal colour band as per 6.6.1(a) shall be sufficiently to identify the pipette as to its nominal volumetric with Fig. 2 and the colour of the band shall be Table 1. 6.6.3 Other ring marks of 6.6.1(b) shall be sufficiently deposi!eci on glass to enable the setting of meniscus and thickness shall be in accordance with Fig. 2 and these ring mark(s) shall be only in black colour. 6.6.4 applied 6.7 Heparin coated pipette shall to the glass at location specified have red or purple colour in Fig. 2. band- Reading and Setting the Meniscus for Type III is made on the lowest point 6.7.1 Reading Liquid Meniscus - Reading of the meniscus. In order that the lowest point may be observed, it is necessary to place a shade of some dark material immediately below and behind the meniscus, which renders the profile of the meniscus dark and clearly visible against a light background. 5 IS : 11383 - 1985 TABLE 1 COLOUR OF BAND OF PIPETTE ( Clause6.6.2 ) NOMINAL CAPACITY COLOTJR CODE 5 10 20 25 44-7 50 100 200 White Orange Black 2 White Green Green Blue Red 6.7.2 Setting a Liquid Meniscus - The setting of the meniscus shall be performed by the methods detailed below. Wherever practicable, the meniscus should descend the position of setting: 4 Method 1 - The position of the lowest point of the meniscus with reference to the graduation line is horizontally tangent to The position of plane of the upper edge of the graduation line. the meniscus is obtained by having the eye in the same plane of the upper edge of the graduation line. The position of the highest point of the meniscus with reference to the graduation line is such that it is in the plane of the middle of the graduation line. The position of the meniscus is obtained by making the setting in the centre of the ellipse formed by the graduation line on the front and the back of the pipette as observed by having the eye slightly above The setting is accurate if the eye the plane of graduation line. is lowered and the ellipse narrows, the highest point of the meniscus remains midway between the front and rear portions of the graduation line. By this method, it is possible to observe the approach of the meniscus from either above or below the line to its proper setting. difference between meniscus positions resulting from the above methods of adjustment is the volume equivalent of one half the thickness of the graduation line. The difference between the two methods of adjustment is unlikely to exceed 0.4 percent volumetric error from stated capacity and a correction can be calculated where necessary. b) Method 2 - 0) The 6.8 Heparin Coating - Heparin costed pip:tte inner surface shall be evenly coated with either sodium, lithium or ammonium as required for end use either entirely or up to calibration line. 6 IS :11383 - 1985 6.8.1 When entire inner surface is coated with sodium or lithium heparin minimum of 5 units and maximum of 10 units of heparin activity shall be present. 6.8.2 When inner surface is coated with sodium heparin up to calibration line a minimum of 2 units and maximum of 5 units of heparin activity shall be present. 6.8.3 When entire inner surface is coated of Type II or calibration line of Type III with ammonium heparin a minimum of heparin activity shall be present. 6.8.4 Heparinized present with drying the functional use. pipette may appear cloudy. of heparin within a capillary up to 2 units This phenomena is and shall not effect of the sheep plasma or human blood 6.8.5 Fluidity - Coagulation shall not be evident when viewed under normal room lighting and tested in accordance with 8.3. 7. DIMENSIONS 7.1 Dimensions and Fig. 1. of Type I pipette shall be in accordance with Table 2 TABLE LENGTH mm 32 75 90 100 f 0.5 mm 2 DIMENSIONS DIAMETER OF TYPE I WALLTHICKNESS OUTSIDE mm 0.8 f 1.4 * 0.2 0.30 mm 0.20 + 0.03 0.20 + 0.03 0.20 + 0.03 0.20 + 0.03 0'02 0.02 0.02 0.02 1.4 & 0.30 1.4 f 0'30 7.2 Nominal capacities with Table 3 and Fig. and dimensions 1. of Type II shall be in accordance 7 \ CHIP NOT TO ENTER BORE 7 --TOE NAIL 0'5max. CHIP 1.5 max. FIQ. 1 PIPETTES, TYPES 1 AND 2 IS : 11383- 1985 TABLE 3 NOMINAL CAPACITIES AND DIMENSIONS OF TYPE II ( Clause7.2 ) NOMINAL CAPACITY MINIJ~UM OUTSIDE DIAMETER MINIMUM WALL THICKNESS MAXIMUM VOLUMETRIC DEVIATION ___--h----~ Accuracy Coefficient Variations & Percent 2.0 I.5 1.5 1'5 1'5 ::; 1.2 I.2 Pl mm f Percent 1.5 ;:2" 1.2 1.2 1.2 1.2 dimensions 7.3 Nominal capacities and accordance with Table 4 and Fig. 2. TABLE NOXINAL CAPACITY IL1 of Type III shall be in 4 NOMINAL GRADUATION MARK(S) AT Erl CAPACITIES MINIMUM OUTSIDE DIAYETER AND DIMENSIONS OF TYPE III VOLUMETRIC EVIATION Coefficient Variation Percent D mm MINIMUM WALL THICKNESS mm MAXIIA;U ____---_-_~ Accuracy TPercent MAY BE FIREPOLISHED BLACK RING MARK 0.5rnax. 7 COLOUR BAND 2 - 6 COLOUR BANDS All dimensions in millimetres. FIG. 2 PIPETTES, TYPE 3 IS : 11383 - 1985 8. TESTS 8.1 Accuracy of Dimensions - of pipettes shall be in accordance Accuracy of dimensions with the relevant tables. for all types 8.1.1 Uniformity of Inside Diameter - The bore of the pipette shall be uniform within the given diameter in relevant table and shall not vary in excess of 0.025 mm in the total length. 8.1.2 Uniformity of Outside Diameter - The outside diameter shall be uniform within the given diameters in relevant tables and tapering in itotal length of the pipette shall not be in excess of 0.02 mm. .8.2 Maximum 8.2.1 Volumetric Deviation Volumetric AccuraT of variation shall not exceed The volumetric accuracy and coefficient the limits given in the respective tables. definition of volumetric accuracy is exDressed as the exoected NOTE -The distribution of mesn volumes around the normal v&me. The definition ofcoefficient of variation is ex?rer;ed as the expected distribution of individual volumes around the normal volume. 8.2.2 Capacity Testfor Type II - A dry pipette and a vesse1 of distilled water shall be allowed to stand at room temperature of 27°C for two hours, The pipette shall be weighed dry and weight indication recorded. -The same pipette shall then be filled with distilled water, by capillary action with specific care to remove all water from the ext.erior of pipette with dry cloth or gauge. The pipette with water content, shall then be reweighed and the weight indication recorded. The recorded weight indication for the dry pipette shall be subtracted from the recorded weight indication, for the pipette filled with distilled water. The difference is representing the balance weight indication reading for the contained water. 8.2.3 Capacity Test for Type III - A dry pipette and a vessel of distilled water shall be allowed to stand at room temperature of 27°C The pipette shall be weighed dry and weight indication for two hours. The same pipette shall then be filled with distilled water, by recorded. XcapiIlary action aI4 a Ijuited to the ring mark in accordance with 6.7 with specific care to remove all water from the exterior of pipette with The pipette with water content, shall then be dry cloth or gauge. reweighed and the weight indication recorded. The recorded weight indication for the dry pipette shall be substracted from the recorded weight indication of the pipette filled with distilled water. The difference is representing the balance weight indication for the contained water. represents Thz 511anc: weight readin: thus determined as per 8.2.2 and 8.2.3 the mass, not corrected for air buoyancy, which is unnscenary in these particular cdses. NOTE 2 - To accurately perform the test of 8.?.2 and 8.2.3 the reliability of the weighing instrument should be confirmed agCn;t a known standard and weighing instrument discrepancy shall not exceed as follows: ~ofmal Capacity of Pipette 1 to 5 cl1 10 to 250 CL1 Discrepancy 0901 mg or better O-01 mg or better NOTE 1 - 11 IS : 11383- 1985 8.2.4 Calculation weighing of Volume - The volume `V' of the is calculated in accordance with IS : 8897-1978*. pipette from, 8.2.5 Capacity Deviation for Single Pipette - In accordance with testing methods outlined in 8.2.2 and 8.2.3 the capacity deviation shall be ascertained as follows: Capacity where V = capacity nominal at reference capacity temperature, and VI = of pipette. deviations, percent = ( VVI) Vl x 100 8.2.6 Capacity Deviations for a iklinimum of 30 Pitettes - Thirty pipettes. taken at random from manufactured lot, shall be tested in accordance with 8.2.2 and 8.2.3. The volumetric deviation from the 30 pipettes shall be ascertained as follows as per accuracy percent and coefficient of variations: 100 x ( F-v, ) Accuracy, percent = Vl where F = mean of sample capacity measurements, of the pipette. 100s +and V- nominal Coejicient of variations Coefficient where of variation, percent = S- 2( J where V v,- tz- vj2 1 sample of pipettes measurement, measurement, measured. by the standard deviation s and VI = individual = mean number 12 z Alternative method Precision: Precision s= where x1 = individual *Tables for calibration of samples can be measured Xl - II 2 ( 32 1 value obtained, of volumetric glassware. n- and method of verification 12 IS : 11383 - 1985 1 = mean value ~~ 2x1 n , and n = number of values obtaine'd. deviation of The variation coefficient ( V percent ) is the standard the mean value expressed in percent can be calculated as: V, percent -= -`T Accuracy: The accuracy may be determined by the deviation arithmetical mean value ( actual value ) from the specified value d=x--0 ( po =- specified A comparison is obtained abbreviated to R percent. R, percent = ( f /&o) ],by 100 _ the d.100 percentage of of the ( d). value ). deviations po IJo It is possible to define both precision and accuracy by mathematical characteristics, provided that distribution is normal, that is, the curve graphically representing the values is a bell shaped curve. 8.3 Fluidity Test for Heparinized shall be tested by one of the following 8.3.1 Sheep Plasma Test - The preparing recalcified sheep plasma a) Sheep plasma Pharmacopoeia b) Pipettes methods. Heparinized pipettes by test shall be conducted by the following process: in accordance heparin ), initially with shall be prepared ( assay for sodium Indian 10 millilitres ( 10 ml ) of added to 2'0 millilitres chloride solution used in calcium chloride solution the prepared sheep plasma shall be ( 2 ml ) of the one percent calcium The sheep plasma and heparin assay. shall be mixed well. 8.3.2 Controls - Sample of both the plain sheep plasma and calcified sheep plasma shall be used as controls in accordance with the following: a) Positive control with recalcified b) flegative control sheep plasma. - A plain ( non-heparinized sheep plasma. A heparinized pipette ) pipette shall be filled with plain shall be filled Immediately after the preparation of the recalcified sheep plasma, the pipette shall be filled by immersing the tips in the recalcified sheep plasma and holding the pipette at such an angle as to facilitate quick filling. The pipette shall be filled to within 5 mm ( in the calibrated pipette from datum mark end ) from the other end and placed in a horizontal position. At the end of one hour period, the pipettes containing plasma 13 IS:11383 - 1985 shall be inspected for evidence of coagulation by carefully snapping off segments of pipettes in approximately 25 mm lengths, and placed on a flat surface ( use a dark background to facilitate observation and composing with control sample ). Coagulation has occurred if the sheep plasma become opaque or if a fine fribin thread is noted. 8.3.3 lieu of known used as Human Whole Blood Test - Human whole blood may be used in sheep plasma by following the steps outlined in 8.3.2. Blood of. donor that does not have clotting mechanism deficiencies shall bea control. 8.4 Heparin Content Test - The heparin content in the pipette shalt be determined by the method for assaying heparin in Indian Pharmacopoeia or other acceptable methodology that will correlate and provide equivalent test results. The result obtained shall represent the heparin content on the inner surface of the pipettes. No heparin from the outside surface of the pipettes shall enter the test sample. 8.5 Resisting to Centrifugal Test - Pipettes which are claimed to be used with centrifugal machine shall be such that no breakage results when pipettes are tested as follows. 8.5.1 The pipettes shall be filled to capacity with distilled water and The centrifuge shall be sealed and suspended or set in centrifuge. accelerated gradually to a speed of 12 000 rev/min. The centrifuge shall run at this speed for 4 minutes only and then allowed to stop without using the brake. 9. REUSE OF PIPETTES may 9.1 Heparinized be reused pipettes are for single use only. Other pipettes by cleaning them with two possible methods given below: A4ethod I - After the pipette is used, insert the pipette into the conical aperture of the rubber stopper on the pipetting aid such that the end of the pipette to emerge at the other end. There is ventilation aperture in the rubber cap to facilitate un-impaired air circulation and the pipette is thus automatically re-filled by capillary action. Once the pipette is full from end to end, empty the pipette by closing the air vent with finger and nipping the rubber cap. The process is repeated for flushing out, using a diluent, a reagent or a sterilizing fluid. It may be necessary to rinse out pipette several times when higher-viscosity liquids are used. Instead of rubber long rubber tube blow out. stopper being used it can be done with sufficient and mouth piece ( known as pipetting aid ) for- 14 IS : 11383 - 1985 Method 2 - Pipette is held between thumb and forefinger ( or with a pair of tweezers ) and allowed to be filled up by capillary action. It is then emptied by putting in into a stoppered test tube. This stopper tube contains either a diluent, a reagent or a sterilizing fluid. The vigorous shaking of the tube The tube is closed with stopper. will rinse the pipette. 10. INSCRIPTIONS 10.1 The following container package: AND MARKINGS shall be marked on the pipette unit inscription a) Dimensions where capacity is not an adjunct or a number indicating the nominal capacity, adjacent -or subject to this number, the symbol `pl' to indicate the unit in terms of which the pipette is graduated. b) 4 4 The inscription temperature. ` 27°C ' to indicate the standard reference For heparinized pipettes a lot or control number shall be indicated and this number shall be traceable to the origin of raw material purchases of the manufacturer record. A statement on expected haparin salt and expected units of heparin with an expiry date ( regarding potency of heparin ) claimed by the manufacturer ). name or trade-mark. accuracy of pipettes and coefficient may also of variation. of the pipettes. be marked with the ISI used in fabrication The manufacturer e> f>Actual figures for 9) Type of glass material 10.1.1 The Certification .package Mark. NOTE - The use of the IS1 Certification Mark is governed bv the orovisions of the Indian Standards Institution ( Certification Marks) Act and the Rules and Regulations made thereunder. The IS1 Mark on products covered by an Indian Standard conveys the assurance that they have been produced to comply with the requirements of that standard under a well-defined system of inspection, testing and quality control which is devised and supervised by IS1 and operated by the producer. IS1 marked products are also continuously checked by IS1 for conformity to that standard as a further safeguard. Details of conditions, under which a licence for the use of the IS1 Certification Mark may be granted to manufacturers or processors, may be obtained from the Indian Standards Institution. 11. PACKING 11.1 The pipettes and the supplier. may be packed as agreed to between the purchaser 15 INTERNATIONAL Base Units QUANTITY SYSTEM UNIT metre kilogram second ampere kelvin candela mole OF UNITS ( SI UNITS ) SYYIBOL m kg A K cd mol Length Mass Time Electric current Thermodynamic temperature Luminous intensity Amount of substance Supplementary QUANTITY Plane angle Solid angle Derived Units QUANTITY Force Energy Power Flux Flux density Frequency Electric conductance Electromotive Pressure, stress force Units UNIT radian steradian SYMBOL rad sr UNIT newton joule watt weber tesla hertz siemens volt Pascal SYMBOL N 1 1 1 1 1 V Pa 1 DJCFINWION N = 1 kg.m/sa J = 1 N.m W=lJ/s T = 1 Wb/ms S = 1 A/V V = 1 W/A w Wh T HZ 1 Wh = 1 V.s 1 Hz = 1 c/s (s-l) 1 Pa = 1 N/ma